Technical Field
The present invention concerns methods and miniaturised apparatus for the processing/manipulation of particles. The invention is applied principally in the implementation of biological protocols on cell samples in reduced volumes, as is often required by a miniaturised analysis approach.
State of the Art
In biology, the centrifuge is one of the instruments most used for the processing of cell samples, in particular with reference to certain stages of the process such as washing the cells. This is necessary for example when starting from a culture, for which it is desirable to remove the culture medium and re-suspend the cells in a saline solution, or when, after an incubation phase with antibodies and/or other reagents, one wants to wash away the excess antibodies and re-suspend the cells in a saline buffer, etc. The potential advantage that can be achieved with the reduction of the volumes involved has led to the miniaturisation of these stages of the process, which has stimulated the development of various methods of processing/manipulation.
In Seger et al. Cell immersion and cell dipping in microfluidic devices, LabChip, 2004, 4, 148-151, pressure-controlled liquid flows are used to exchange cells from one suspension buffer to another (washing), or to immerse cells originally present in a first buffer in a second buffer for a controlled time (incubation or reagent-sampling). The disadvantage of this approach is that it is necessary to maintain a controlled flow of the various liquids involved. This implies a greater consumption of reagents and a greater complexity of the system, linked with the necessity of producing pressure to flush (move) the liquids.
A similar approach is used by EVOTEC to submit single cells trapped in dielectrophoresis cages to particular reagents, but here too it is necessary to have controlled flows of reagents.
The patent application PCT/WO 00/69565 a G. Medoro describes an apparatus and a method for the manipulation and identification of particles making use of closed cages with dielectrophoretic potential. The method described teaches how to control the position of each particle independently of all the others in a two-dimensional space. The force used to trap the particles in suspension is negative dielectrophoresis. The individual control of the manipulating operations is achieved by the programming of memory elements and circuits associated with each element of an array of electrodes and sensors integrated in the same substratum.
The U.S. Pat. No. 6,294,063 Becker et al. describes a method and apparatus for the manipulation of packages of solid, liquid or gaseous biological material by means of a distribution of programmable forces.
In the application for an Italian patent BO2005A000481, Medoro et al., some methods are given for manipulating particles with arrays of electrodes, and some methods and apparatus for identifying them, which are however similar to those of the already mentioned patent PCT/WO 00/69565.
Although it does not allow a precise control of the position of the particles, it is also possible to use Travelling Wave dielectrophoresis to shift the particles.
In other cases electrodes are not necessary to manipulate particles situated inside the microchambers.
Then there are many known methods of generating optical traps (optical traps, optical tweezers), which are based on the differences of the refraction index of the particles with respect to the suspension medium (for example A. Ashkin et al, Optical trapping and manipulation of single living cells using infrared laser beams, Nature, 330(6150) (1987) 769.)
In this case the particles are typically trapped in optical intensity maximums, generated for example by focussing a laser beam through the lens of a microscope. The manipulation of a multiplicity of particles may be obtained with various optical methods in the prior art, based on similar principles, for example using arrays of VCSEL (Vertical Cavity Surface Emitting Laser), or using holographic optical traps.
Other techniques are known, which combine the projection of images with luminous intensity gradients, the use of an electric field in the liquid and a device with a photosensitive substratum, to create so-called Opto-Electronic cages (for example U.S. Pat. No. 6,387,707B1 assigned to Seul et Al., or Pei Yu Chiou et al, “Massively parallel manipulation of single cells and microparticles using optical images”, Nature Vol 436, 21 Jul. 2005, pp 370-372). In practice a dielectrophoretic field is realised, controlled non by the shape of the electrodes but by the image projected on the photosensitive substratum. In this way it is therefore possible to manipulate the particles present in the liquid.
Lastly other techniques are known for moving particles. In S. Gaugiran, et al., “Optical manipulation of microparticles and cells on silicon nitride waveguides,” Opt. Express 13, 6956-6963 (2005), some particles (artificial or biological such as yeasts and cells) are pushed along wave guides be the radiation pressure of the evanescent field of a laser which is spread inside the guide itself.
The international patent application in the name of the same Applicant, filed on 22 Mar. 2006 and concerning a method and an apparatus for making the characterisation and/or the count of particles of any type, for example biological particles such as cells or their parts, in which the control of the position of each particle present in a sample is used in order to shift those particles in a deterministic or statistical way, to detect their presence and/or characterise the type with optical integrated and/or impedenziometric sensors. In particular non uniform fields of force are used, variable times and optical or impedenziometric sensors located under or close to an array of electrodes, integrated with them in a single chip. The fields of force may have positive or negative dielectrophoresis, electrophoresis or electro-hydrodynamic movements, characterised by a set of stable points of equilibrium.
However, among the methods reported in the above-mentioned inventions concerning micromanipulators of cells/particles based on dielectrophoresis or other optical or opto-electronic techniques, although a flow is not necessary, no method is contemplated for allowing those typical operations such as washing and/or incubation of the cells or particles, as is necessary in many biological protocols.